ABSTRACT Background: Heart valve disease affects millions of people worldwide due to an increase in life expectancy in industrialized countries, persistence of rheumatic fever in developing countries, and congenital heart defects. Heart valve disease is currently treated with valve replacement, which often results in complications and is not a suitable option for pediatric patients. To address this issue, tissue engineered heart valves (TEHV) can be developed to decrease the risks of complications and remove the need for multiple surgeries through their ability to grow and remodel. Despite advances in TEHV, one of the challenges is finding a suitable cell source for TEHV scaffolds. The other challenge is matching the anisotropic microstructure and the mechanical properties of the native valve. Here, we propose to replicate the microarchitecture and heterogeneity of the leaflet layers using 3D bioprinting in order to generate a valve with better mechanical properties, anisotropy, and biological integration. The overall hypothesis is we can use generated valvular interstitial cells (VICs)-like cells to create a living TEHV that mimics the structural and mechanical properties of native valves. Approach: We propose to differentiate human induced pluripotent stem cells (iPSCs) into VIC-like cells using both biochemical and mechanical cues. iPSCs will be differentiated into mesenchymal stem cell (MSC)-like cells using our feeder-free differentiation protocol. Cells will then be encapsulated in polyethylene (glycol) diacrylate (PEGDA) hydrogels, and grafted with adhesion peptide (RGDS). We hypothesize VIC-like cells can be differentiated from iPSCs using our feeder-free protocol and 3D cell culture environment. We will determine the efficiency of the differentiation using flow cytometry to stain for MSC-like surface markers and characterize VIC- like phenotype by the expression of ?SMA, vimentin, F-actin, and ECM production. In addition, we propose to develop a TEHV with remodeling potential that mimics the structural and mechanical properties of a native heart valve. Using 3D bioprinting, we will print layers of polycaprolactone (PCL) and layers of PEGDA containing VIC- like cells to generate the 3D structure. The inner structure will control the anisotropic properties of the valve. We hypothesize the inner structure and the heterogeneity between the bioprinted layers will result in similar mechanical properties of native valve leaflets and that VIC-like cells will enable remodeling of the scaffold.!To characterize our scaffold, the elastic modulus, swelling ratio, average molecular weight, and cell viability will be determined. In addition, we also seek to use CT scans to generate patient-specific reconstructed 3D aortas that can be 3D printed with the leaflet microstructures. Expected Results: We plan to address the need for a more functional TEHV. Using our feeder-free approach, we expect to generate VIC-like cells from iPSCs. By mimicking the microstructure of the valve leaflet using biomaterials, we expect to generate a valve with better mechanical properties and biological integration.